Also untested, since I don't have Bio::SeqIO:

use Bio::SeqIO;
use strict;
use warnings;

my @V;
open (LIST1, 'list') || die;
while (<LIST1>){
    push @V, (split(/\t/, $_))[0];
}
close(LIST1);

my $seqIO_object = Bio::SeqIO->new(-file => 'infile.gb');
my $seq_object = $seqIO_object->next_seq;

for my $feat_object ($seq_object->get_SeqFeatures) {
    if ($feat_object->primary_tag eq 'CDS' &&
        $feat_object->has_tag('locus_tag')) {
        for my $V3 ($feat_object->get_tag_values('locus_tag')) {
            for my $V1 (@V) {
                print "locus_tag $V1 is unique\n"
                    if $V1 eq $V3;
            }
        } 
    }
}
[download]

I could probably streamline this further if I knew exactly what you were trying to do, but oh well.